The long term objectives of this project are to identify beta-cell surface autoantigens and to determine their role in the specific loss of islet beta cells. Our hypothesis is that beta-cell specific surface antigens guide the immune-associated beta-cell attack. The major autoantigen detected by immunoprecipitation is a Mr 64,000 protein, identified as a hydrophobic isoform of glutamic acid decarboxylase (GAD). A human islet expression CDNA library was used to clone and sequence full-length GAD2 CDNA, corresponding to a 5.6-kb MRNA in both islets and brain. The deduced 585- amino acid sequence of GAD2, corresponding to a protein of about 65K, shows <65% identity to conserved GAD1 brain sequence which show .96% deduced amino acid sequence identity among rat, mouse, cat and man. The GAD2 CDNA- directed in vitro transcription and translation produced a p64 product which was immunoprecipitated by IDDM, but not control sera as well as by p64 GAD2 specific monoclonal antibody. The successful molecular cloning and determination of the primary structure of the 64K antigen, p64GAD2, is of major significance for diabetes research. The IDDM auto-epitope of p64GAD2 will be analyzed to better define the role of this autoantigen in the etiology and pathogenesis of IDDM. GAD isoforms important in several autoimmune diseases, including Stiff-Man syndrome, may be explained in part by gene duplication. We confirm the mapping of GAD1 (3.7-kb transcript) to chromosome 2, and demonstrate the GAD2 maps to chromosome 10 and a third transcript (2.5-kb) (GAD3) to chromosome 22. The revised application has four specific aims: 1) To test the hypothesis that p64GAD2 is a unique autoantigen in IDDM to explain the specific loss of beta-cells. The GAD2 CDNA sequence will be compared to that of human islet GAD1 and GAD3 CDNA. The islet GAD2 CDNA from monkey, dog, rat and mouse will be compared since they show a decreasing order of IDDM serum reactivity with their 64K antigen. The genomic GAD2 structure will be determined to explain differences between islet and brain primary structure possibly induced by alternative splicing. 2) To test the hypothesis that unique auto-epitope(s) in p64GAD2 are important to the development of IDDM. Specifically, in vitro mutagenesis of GAD2 CDNA and overlapping synthetic polypeptides will be used to map the auto-epitope(s). 3) To test the hypothesis that aberrant presentation of p64GAD2 is important in the pathogenesis of IDDM. The structural elements of GAD which determine membrane association and condiitons inducing cell surface expression will be detemined. 4) To test the hypothesis that p64GAD2 and antibodies to beta-cell specific islet cell surface antigens mark active beta-cell autoimmunity and predict IDDM. Recombinant p64GAD2 will be used in radioligand binding assays for GAD autoantibodies in a) a population-based case-control study of new onset IDDM children and controls, b) the Seattle prospective family study, and c) the Diabetes Prediction Study.